Fig. 5: Inhibition of GJ communication increases aldosterone production.
a, GJA1 expressed in subcapsular non-ZF cells. Human adrenal section stained with mouse anti-CYP17A1 (green) and rabbit anti-GJA1 (red) antibodies. AGJ (yellow arrowhead) is present in subcapsular cells not expressing the ZF marker CYP17A1. This region immunostain for DAB2 but not for CYP11B2 (white box in Supplementary Fig. 6a). Dashed line demarcates border with capsule. Left image, ×63 magnification; right image, ×100 magnification. b, Connexin mimetic peptide Gap27 increases CYP11B2 expression and aldosterone production in Ang II-stimulated H295R cells. CYP11B2 (n = 10) and aldosterone (n = 12 except for 250 μM Gap27, n = 11) are increased in stimulated H295R cells treated with Gap27, which selectively blocks GJ communications. Results expressed as fold change relative to cells treated with 0 μM Gap27. The effect of Gap27 on unstimulated cells is shown in Supplementary Fig. 8a. Ten percent dimethyl sulfoxide (DMSO 10%) treatment on unstimulated cells (n = 8) was used as control for enhanced cell membrane permeability. Statistical significance measured using the Kruskal–Wallis H test; CYP11B2, χ2(4) = 43.03, P = 1.02 × 10−8 and aldosterone, χ2(4) = 42.25, P = 1.48 × 10−8, respectively. Post hoc testing was performed using Dunn’s multiple comparison test (compared to 0 μM Gap27). For CYP11B2, #P = 0.0039, ##P = 0.002. For aldosterone, *P = 0.0310, ***P = 0.0005, ****P < 0.0001. c, Silencing of GJ increases CYP11B2 expression and aldosterone production. CYP11B2 (n = 10) and aldosterone (n = 17) is increased in H295R cells with decreased GJ communications due to cosilencing of the genes GJA1 and GJC1 (SiGJA1/GJC1) compared to the silenced scramble RNA control (SiScr). GJA1 and GJC1 mRNA and protein expression is shown in Supplementary Fig. 8e,f. Results expressed as fold change relative to SiScr cells. Statistical analysis performed using two-sided Student’s t-test. **P = 0.0097, ****P < 0.0001. d, Gap27 increases CYP11B2 expression and aldosterone production in Ang II-stimulated primary adrenal cells. CYP11B2 (n = 10) and aldosterone (n = 10) are increased in stimulated primary adrenal cells treated with Gap27. Effect of Gap27 in unstimulated primary adrenal cells is shown in Supplementary Fig. 8g. mRNA expression was normalized by β-actin. Results expressed as fold change relative to Ang II-stimulated cells. Statistical analysis was performed using two-sided Student’s t-test. ****P = 0.0005, *P = 0.0438. Data are presented as mean ± s.e.m. n = biological independent replicates from three independent experiments. The statistics used to produce these plots are provided as Source Data Fig. 5.
